Lipidomics Study of Huashi Baidu Granules in the Treatment of Respiratory Syncytial Virus Pneumonia Model Mice
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摘要:
目的 探究化湿败毒颗粒对呼吸道合胞病毒(Respiratory syncytial virus, RSV)肺炎模型小鼠的治疗作用以及对肺组织脂质代谢的影响。 方法 将BALB/c小鼠随机分为空白组、模型组、利巴韦林组、地塞米松组、化湿败毒颗粒低剂量组和化湿败毒颗粒高剂量组。分别采集各组小鼠肺组织样本, 观察其病理改变, 检测肺组织中RSV-F、RSV-G、IL-1β、IL-6、TNF-α mRNA表达水平。采用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱联用(UPLC-Q Exactive Orbitrap MS)技术对肺组织进行脂质组学分析, 寻找脂质代谢变化。 结果 与空白组相比, 模型组小鼠肺组织出现肺间质增厚, 可见明显炎症浸润。肺组织病毒载量及肺部炎症因子如IL-1β、TNF-α mRNA水平显著升高(P＜0.001)。同时, 与空白组相比, 模型组呈现脂质代谢的明显异常, 具体表现为磷脂酰丝氨酸(PS)、磷脂酰甘油(PG)、鞘磷脂(SM)、神经酰胺(Cer)、甘油三酯(TG)、甘油二酯(DG)、脂肪酸(FA)、醚磷脂酰胆碱(PC O)、醚溶血磷脂酰胆碱(LPC O)等脂质代谢紊乱。经化湿败毒颗粒(25.12 g·kg-1·d-1)干预可以调节上述脂质紊乱。 结论 化湿败毒颗粒可以通过调控脂质代谢, 降低RSV感染小鼠肺组织中的病毒载量, 减轻炎症反应等途径治疗RSV肺炎。 Abstract:OBJECTIVE To investigate the therapeutic effect of Huashi Baidu Granules on respiratory syncytial virus (RSV) pneumonia model mice and its effect on lung lipid metabolism. METHODS BALB/c mice were randomly divided into control group, model group, Ribavirin group, Dexamethasone group, low dose group of Huashi Baidu Granules and high dose group of Huashi Baidu Granules. Lung tissue samples of mice in each group were collected to observe pathological changes and detect mRNA expression levels of RSV-F, RSV-G, IL-1β, IL-6 and TNF-α genes. The lipid components of lung tissue were extracted and analyzed by ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UPLC-Q Exactive Orbitrap MS) to find the changes in lipid metabolism. RESULTS The model group exhibited pulmonary interstitial thickening and inflammatory infiltration compared to the control group, the mRNA levels of viral load and inflammatory factors in lung tissue such as IL-1β and TNF-α were significantly increased (P < 0.001). Moreover, the model group exhibited significant abnormalities in lipid metabolism, including disorders in phosphatidylserine (PS), phosphatidylglycerol (PG), sphingomyelin (SM), ceramide (Cer), triglyceride (TG), diglyceride (DG), fatty acid (FA), ether phosphatidylcholine (PC O), ether lysophosphatidylcholine (LPC O). However, after the treatment of Huashi Baidu Granules (25.12 g·kg-1·d-1), the above indexes showed a significant improvement. CONCLUSION Huashi Baidu Granules can treat RSV pneumonia by regulating lipid metabolism, reducing viral load and alleviating inflammatory response in lung tissue. -
Key words:
- respiratory syncytial virus /
- pneumonia /
- Huashi Baidu Granules /
- lipidomics
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图 1 各组小鼠肺组织HE染色结果(×200)
注: 与空白组比较, ###P＜0.001;与模型组比较, *P＜0.05, * *P＜0.01, * * *P＜0.001。x±s, n=4。
Figure 1. HE staining results of lung tissue in each group of mice(×200)
图 2 各组小鼠病毒载量及炎症因子基因表达水平
注: 与空白组比较, ###P＜0.001;与模型组比较，*P＜0.05, * * *P＜0.001。x±s, n=6。
Figure 2. Gene expression levels of viral load and inflammatory factors in each group of mice
图 4 小鼠肺组织脂质鉴定分析
注: A.正负离子模式下RSV肺炎小鼠肺组织中检测到的脂质旭日图; B.正离子模式下各组样本的PCA图; C.负离子模式下各组样本的PCA图。
Figure 4. Identification and analysis of lipid in mouse lung tissue
图 5 小鼠肺组织差异脂质分析
注: A.正离子模式下小鼠肺组织差异脂质热图; B.负离子模式下小鼠肺组织差异脂质热图; C.差异表达脂质亚类盒须图; D.与空白组比较，模型组差异脂质富集分析图；E.与模型组比较，化湿败毒颗粒低剂量组差异脂质富集分析图。D~E图中，红色代表脂质类别呈上调趋势，蓝色代表脂质类别呈下调趋势，紫色代表该脂质类别既有上调脂质又有下调脂质，气泡大小与脂质数量呈正相关。与空白组比较, #P＜0.05, ##P＜0.01, ###P＜0.001;与模型组比较, *P＜0.05, * *P＜0.01。x±s, n=9。
Figure 5. Differential lipid analysis of mouse lung tissue
图 6 差异脂质互作分析
注: A.正离子模式下差异脂质互作图(35个节点, 366条边); B.负离子模式下差异脂质互作图(7个节点, 11条边)。通过P＜0.001和Spearman相关系数r>0.75或r＜-0.75筛选。
Figure 6. Differential lipid interaction analysis
图 7 化湿败毒颗粒调节肺脂质改善RSV肺炎
Figure 7. Huashi Baidu granules improve RSV pneumonia through regulating of lung lipids
表 1 qPCR引物序列
Table 1. qPCR primer sequence
引物名称 引物序列(5′→3′) GAPDH-F AGGTCGGTGTGAACGGATTTG GAPDH-R TGTAGACCATGTAGTTGAGGTCA IL-1β-F GCAACTGTTCCTGAACTCAACT IL-1β-R ATCTTTTGGGGTCCGTCAACT IL-6-F TAGTCCTTCCTACCCCAATTTCC IL-6-R TTGGTCCTTAGCCACTCCTTC TNF-α-F CCCTCACACTCAGATCATCTTCT TNF-α-R GCTACGACGTGGGCTACAG RSV-F-F AACAGATGTAAGCAGCTCCGTTATC RSV-F-R GATTTTTATTGGATGCTGTACATTT RSV-G-F CGGCAAACCACAAAGTCACA RSV-G-R TTCTTGATCTGGCTTGTTGCA 
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表 2 正负离子模式肺组织差异脂质
Table 2. Lung differential lipids in positive and negative ion mode
差异脂质 保留时间/min 质荷比m/z 加和离子 化学结构式 模型组/空白组 化湿败毒颗粒高剂量组/模型组 FC P值 FC P值 DG 50∶0 12.46 843.78 [M+Na]+ C53H104O5 3.28 2.46E-06 0.54 9.75E-03 DG 52∶4 11.69 863.74 [M+Na]+ C55H100O5 2.00 7.54E-04 0.73 3.19E-02 HexCer 42∶2;2O|
HexCer 18∶1;2O/24∶17.71 810.67 [M+H]+ C48H91NO8 1.49 2.25E-02 0.69 3.42E-02 LPC O-16∶0 2.25 482.36 [M+H]+ C24H52NO6P 4.04 1.69E-07 0.66 1.41E-02 PC 38∶5 5.93 808.59 [M+H]+ C46H82NO8P 1.56 3.49E-02 1.52 3.34E-04 PC 40∶4 7.09 838.63 [M+H]+ C48H88NO8P 1.65 1.62E-05 0.82 4.58E-02 PC 42∶9 5.58 856.58 [M+H]+ C50H82NO8P 1.83 1.67E-02 0.74 4.94E-02 PC O-30∶0 6.35 692.56 [M+H]+ C38H78NO7P 1.57 7.06E-04 0.75 2.39E-02 PC O-36∶4 6.36 768.59 [M+H]+ C44H82NO7P 2.55 1.51E-07 0.69 5.29E-03 PC O-38∶4 6.88 796.63 [M+H]+ C46H86NO7P 1.96 1.50E-06 0.80 4.28E-02 PC O-38∶5 6.37 794.60 [M+H]+ C46H84NO7P 3.48 8.14E-09 0.69 2.61E-02 PC O-38∶6 6.21 792.59 [M+H]+ C46H82NO7P 1.56 1.18E-04 0.79 5.97E-03 PC O-40∶4 7.72 824.66 [M+H]+ C48H90NO7P 2.27 5.29E-05 0.59 2.10E-03 PC O-40∶7 6.20 818.61 [M+H]+ C48H84NO7P 6.21 6.09E-11 0.75 3.90E-02 PC O-40∶8 6.38 816.59 [M+H]+ C48H82NO7P 2.76 1.30E-08 0.73 1.36E-02 PG 36∶2 5.89 792.58 [M+NH4]+ C42H79O10P 2.56 8.47E-07 0.70 7.33E-03 SM 36∶3;3O 7.03 743.57 [M+H]+ C41H79N2O7P 1.88 5.53E-04 0.58 1.36E-02 SM 40∶7;3O 6.36 791.57 [M+H]+ C45H79N2O7P 1.52 2.30E-05 0.78 5.21E-03 TG 54∶6|TG 16∶0_16∶0_22∶6 10.94 901.72 [M+Na]+ C57H98O6 0.36 6.80E-04 2.30 2.50E-03 TG 54∶6|TG 16∶0_18∶2_20∶4 10.75 901.72 [M+Na]+ C57H98O6 0.79 3.51E-02 1.30 2.16E-02 TG 54∶7|TG 16∶0_18∶3_20∶4 10.48 894.76 [M+NH4]+ C57H96O6 0.69 1.04E-02 1.32 3.18E-02 TG 56∶5|TG 16∶0_18∶1_22∶4 11.43 931.77 [M+Na]+ C59H104O6 0.68 7.97E-04 1.30 2.10E-02 TG 56∶5|TG 18∶0_18∶1_20∶4 11.56 926.81 [M+NH4]+ C59H104O6 0.75 1.48E-02 1.46 1.48E-03 TG 56∶5|TG 18∶1_18∶2_20∶2 11.34 926.81 [M+NH4]+ C59H104O6 0.60 2.85E-03 1.44 2.96E-02 TG 56∶6|TG 16∶0_18∶1_22∶5 11.19 924.81 [M+NH4]+ C59H102O6 0.63 2.67E-03 1.32 3.85E-02 TG 56∶6|TG 16∶0_18∶2_22∶4 11.16 929.75 [M+Na]+ C59H102O6 0.75 3.85E-03 1.33 9.00E-03 TG 56∶6|TG 18∶1_18∶1_20∶4 11.16 929.76 [M+Na]+ C59H102O6 0.74 2.81E-03 1.29 2.16E-02 TG 56∶7|TG 16∶0_18∶1_22∶6 10.96 927.75 [M+Na]+ C59H100O6 0.63 1.09E-03 1.33 2.87E-02 TG 56∶7|TG 16∶0_18∶2_22∶5 10.77 922.78 [M+NH4]+ C59H100O6 0.53 4.12E-05 1.37 2.13E-02 TG 56∶8|TG 16∶0_18∶2_22∶6 10.58 920.77 [M+NH4]+ C59H98O6 0.56 8.28E-06 1.40 1.18E-02 TG 56∶8|TG 16∶0_20∶4_20∶4 10.58 925.73 [M+Na]+ C59H98O6 0.59 1.21E-05 1.31 9.27E-03 TG 56∶8|TG 18∶2_18∶2_20∶4 10.38 925.73 [M+Na]+ C59H98O6 0.58 2.09E-05 1.30 3.46E-02 TG 58∶6|TG 18∶0_18∶2_22∶4 11.51 952.83 [M+NH4]+ C61H106O6 0.69 1.53E-03 1.29 2.42E-02 TG 58∶6|TG 18∶1_18∶1_22∶4 11.50 957.79 [M+Na]+ C61H106O6 0.66 5.76E-04 1.27 4.20E-02 TG 58∶7|TG 18∶0_18∶1_22∶6 11.38 950.81 [M+NH4]+ C61H104O6 0.67 9.74E-04 1.61 3.24E-04 TG 58∶7|TG 18∶0_20∶3_20∶4 11.21 950.81 [M+NH4]+ C61H104O6 0.53 2.83E-04 1.37 1.98E-02 TG 58∶8|TG 18∶0_20∶4_20∶4 10.97 953.75 [M+Na]+ C61H102O6 0.69 5.57E-04 1.28 3.91E-02 TG 58∶8|TG 18∶1_18∶2_22∶5 10.76 948.80 [M+NH4]+ C61H102O6 0.44 8.13E-05 1.51 3.22E-02 TG 58∶9|TG 18∶1_18∶2_22∶6 10.60 951.74 [M+Na]+ C61H100O6 0.61 6.06E-05 1.24 4.12E-02 TG 60∶5|TG 24∶1_18∶2_18∶2 12.10 982.88 [M+NH4]+ C63H112O6 0.54 4.80E-03 1.44 3.27E-02 Cer 42∶1;2O|Cer 18∶0;2O/24∶1 8.59 708.65 [M+CH3COO]- C42H83NO3 1.37 3.69E-03 1.28 4.05E-02 FA 18∶3 1.61 277.22 [M-H]- C18H30O2 1.53 2.86E-05 0.79 3.75E-02 FA 24∶2 4.60 363.33 [M-H]- C24H44O2 2.24 1.36E-09 0.77 4.54E-03 PC 30∶0|PC 14∶0_16∶0 5.63 750.53 [M+HCOO]- C38H76NO8P 0.61 2.25E-06 0.79 9.70E-03 PC 36∶3|PC 18∶1_18∶2 5.90 828.58 [M+HCOO]- C44H82NO8P 0.67 2.26E-03 0.76 2.93E-03 PC 36∶4|PC 16∶0_20∶4 5.74 826.56 [M+HCOO]- C44H80NO8P 0.72 5.48E-05 0.79 2.56E-05 PC O-32∶1|PC O-16∶1_16∶0 6.60 762.56 [M+HCOO]- C40H80NO7P 0.45 1.81E-07 0.77 7.73E-04 PS 38∶3|PS 18∶0_20∶3 5.54 812.55 [M-H]- C44H80NO10P 0.81 1.46E-02 1.37 6.34E-03 SM 34∶0;2O 5.73 763.59 [M+CH3COO]- C39H81N2O6P 1.48 3.21E-05 1.25 2.18E-02 SM 34∶2;2O 4.99 759.57 [M+CH3COO]- C39H77N2O6P 1.31 7.49E-06 1.25 3.06E-02
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